Tobacco-specific nitrosamines, the best known of which are N2-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), are widely considered to be among the most important carcinogens in tobacco and tobacco smoke and are rated as carcinogenic to humans by the International Agency for Research on Cancer. Hundreds of millions of smokers and smokeless tobacco users in the world are exposed daily to considerable amounts of these potent carcinogens, which readily induce tumors at relevant sites such as the lung and oral cavity in laboratory animals. This renewal application proposes to extend our studies on metabolism and DNA adduct formation by NNN and NNK in rats, in smokers, and in smokeless tobacco users. These ongoing studies have been supported in the most recent term by a MERIT award. Under the support of this grant, we have elucidated most of the metabolic pathways of NNN and NNK, and have characterized many of the resulting DNA adducts. This research has led to our development of biomarkers of human uptake of NNN and NNK, such as total NNAL, now widely used as a carcinogen biomarker in smokers. We now propose to extend this biomarker development research to the metabolic activation and DNA binding of NNN and NNK in humans. Our overall hypothesis is that these uptake and metabolic activation biomarkers can identify those tobacco users who are at highest risk for cancer, so preventive measures such as intensive cessation therapy and early detection methods can be applied at a young age. While the uptake biomarkers for NNN and NNK are now well established, the metabolic activation and DNA adduct biomarkers, critical on the path to cancer development, have been only superficially examined to date in humans. To address this problem, we propose coordinated studies in F-344 rats, highly susceptible to the carcinogenic effects of NNN and NNK, and in humans. Thus, our specific aims are: 1. Using liquid chromatography - nanoelectrospray ionization - high resolution tandem mass spectrometry (LC-NSI-HRMS/MS), characterize DNA phosphate adducts of metabolically activated NNN and NNK as formed in tissues of F-344 rats treated with these carcinogens. 2. Investigate the formation of DNA adducts resulting from metabolic activation by 5'-hydroxylation of NNN in F-344 rats. 3. Characterize the metabolic activation and DNA binding of [pyridine-D4]NNN in smokeless tobacco users as a basis for effective biomarker development. 4. Using LC-NSI-HRMS/MS, determine the longitudinal stability of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB)-releasing DNA adducts in human oral cell DNA and analyze for specific adducts of NNN, NNK, and NNAL, including those characterized in Specific Aims 1-3. Our goal is to establish these oral cell DNA adducts as biomarkers of tobacco-specific nitrosamine metabolic activation in smokers and smokeless tobacco users.